DAG (Diacylglycerol) Assay Kit

1. DAG Standard : One 1 mL vial of 1 mM 1-2-dioleoyl-sn-glycerol.
2. 10X Assay Buffer : One 1.5 mL vial.
3. Kinase Mixture : Two 1 mL vials containing ATP.
4. Lipase Solution : Three 1.4 mL vials.
5. Enzyme Mixture : Three 1.75 mL vials.
6. Fluorometric Probe : One 110 μL amber vial.

• Suitable for use with cell lysates
• Detection sensitivity of 4uM DAG
• DAG standard included
• NOTE: Each sample replicate requires 2 assays for proper data calculation.

DAG Standard: Thaw at room temperature. Once homogeneous and mixed well, maintain the standard at room temperature during assay preparation. The solution is stable for 1 week at room temperature. For longer term storage, the solution should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws. 1X Assay Buffer: 10X Assay Buffer should be thawed/maintained at 4 °C during assay preparation. Dilute the 10X Assay Buffer with deionized water. Stir to homogeneity. The 1X solution is stable 3 for 1 month at 4 °C. For longer term storage, any unused 10X stock material should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws. Kinase Mixture, Lipase Solution, and Enzyme Mixture: Thaw at 4 °C. Once homogeneous and mixed well, maintain the solution at 4 °C during assay preparation. The solution is stable for 1 week at 4 °C. For longer term storage, the solution should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws. Note: These components are provided in multiple tubes to minimize multiple freeze/thaws. Fluorometric Probe: Thaw and maintain at room temperature during assay preparation. Any unused material should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws. PEU (pre-equilibrated upper phase) Solution: Mix 50 mL of chloroform, 50 mL of methanol, and 45 mL of 1M NaCl in a glass container. Shake or mix the solution well, then allow it to separate into 2 phases. Use the upper phase for washing during the extraction.

Urine, plasma and serum: This kit is not recommended for these samples. Cell lysates: 7 For adherent cells, remove media and wash cells twice with cold PBS. Harvest ~1x 10 cells by using a rubber policeman. Do not use proteolytic enzymes. Centrifuge at 1500 x g for 10 minutes. Carefully remove the supernatant and resuspend in 1 mL of cold PBS. Proceed to step 1 of the extraction procedure below. 4 7 For suspension cells, collect ~1 x 10 cells by centrifugation at 1500 x g for 10 minutes. Carefully remove the supernatant and wash the cell pellet with cold PBS. Repeat PBS wash/centrifugation once more. Carefully discard the supernatant and resuspend in 1 mL of cold PBS. Proceed to step 1 of the extraction procedure below: Extraction Procedure

1. Sonicate the 1 mL of cell suspension on ice.
2. Add 1.5 mL of methanol to the sonicated sample.
3. Add 2.25 mL of 1 M NaCl and 2.5 mL of chloroform to the sample. Vortex well.
4. Centrifuge at 1500 x g for 10 minutes at 4 °C to separate the phases.
5. Carefully remove the upper aqueous phase and discard.
6. Wash the lower chloroform phase 2 times with 2 mL of pre-equilibrated upper phase (PEU) (see Preparation of Reagents Section). Separate the phases each time by centrifuging at 1500 x g for 10 minutes at 4 °C. Carefully remove the upper phase and discard each time.
7. After the final wash, carefully transfer the lower organic phase to a glass vial or tube (a syringe works well). Avoid transferring any remaining upper, aqueous phase.
8. Dry the lower phase in a speedvac or under a gentle stream of nitrogen.
9. Resuspend the dried sample with 50 μL of 1X Assay Buffer (1:20 of the original volume). Samples may be stored at -80 °C for up to a month.

Note: A freshly prepared standard curve should be used each time the assay is performed. Maintain the Kinase Mixture, Lipase Solution, and Enzyme Mixture at 4 °C during assay preparation.

1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate. Note: Each unknown sample replicate requires two paired wells, one to be treated with Kinase Mixture (+Kin) and one without (-Kin) to determine phosphatidic acid background (1X Assay Buffer will be added in place of the Kinase Mixture).
2. Add 20 μL of the DAG standards, samples or blanks to the 96-well microtiter plate.
3. Add 20 μL of Kinase Mixture to the standards and to one half of the paired sample wells, and mix the well contents throughly.
4. Add 20 μL of 1X Assay Buffer to the other half of the paired sample wells and mix thoroughly.
5. Incubate at 37 °C for 1-2 hours.
6. Transfer 20 μL of the mixture to a 96-well plate suitable for fluorescence measurement.
7. Add 40 μL of Lipase Solution to each well.
8. Incubate at 37 °C for 30 minutes.
9. During the step 8 incubation, separately prepare the desired volume of Detection Enzyme Mixture according to Table 2 below, based on the number of tests to be performed. Maintaining 5 all components and mixtures at 4 °C throughout this step, add components in the following sequence: a. In a tube, add the appropriate volume of Enzyme Mixture. b. To the Enzyme Mixture, add the corresponding volume of Fluorometric Probe. Mix well and immediately use. Note: Detection Enzyme Mixture may appear slightly pink in color. This is normal background and should be subtracted from all absorbance values (see step 12 for calculation). Enzyme Fluorometric Total Volume of # of Tests in Mixture (mL) Probe (μL) Detection Enzyme 96-well Plate Mixture (mL) (100 μL/test) 5 50 5.05 100 2.5 25 2.525 50 1.25 13 1.263 25 Table
10. Preparation of Detection Enzyme Mixture
11. Transfer 50 μL of the above Detection Enzyme Mixture (from step 7) to each well.
12. Cover the plate wells to protect the reaction from light.
13. Incubate at room temperature for 10 minutes.
14. Read the plate with a fluorescence microplate reader equipped for excitation in the 530-560 nm range and for emission in the 585-595 nm range. Calculation of Results
    1. Determine the Average Relative Fluorescence Unit (RFU) values for each sample, control, and standard.
    2. Subtract the average zero standard value from itself and all standard values.
    3. Graph the standard curve (see Figure 1).
    4. Subtract the sample well values without Kinase Mixture (-Kin) from the sample well values containing Kinase Mixture (+Kin) to obtain the difference. Net RFU = (RFU+Kin) - (RFU-Kin)